Project Summary This project is focused on understanding of the molecular mechanisms that control energy conversion within the cell power plant, the mitochondrion. Transport of ion and metabolites across the inner mitochondrial membrane is the foundation of mitochondrial physiology. Here we study the molecular mechanisms involved in passive uptake of H+ (?mitochondrial H+ leak?) and Ca2+ (?mitochondrial Ca2+ uptake?) into mitochondria down the negative voltage (??) across the inner mitochondrial membrane. The mitochondrial H+ leak is responsible for the conversion of chemical energy of mitochondrial substrates into heat. The mitochondrial Ca2+ uptake lets Ca2+ enter mitochondria during cytosolic Ca2+ transients to stimulate Ca2+-dependent enzymes of the Krebs cycle and mitochondrial energy conversion. The mitochondrial H+ leak and Ca2+ uptake are mediated by specialized integral proteins of the inner mitochondrial membrane called uncoupling proteins (UCPs) and mitochondrial Ca2+ uniporter (MCU) correspondingly. The molecular and functional characterization of UCPs and MCU has been difficult due to the inability to measure H+ and Ca2+ currents across the inner mitochondrial membrane directly. We have resolved this major technical barrier and developed a method for direct patch- clamp recording of UCP and MCU currents across the whole inner mitochondrial membrane. Using this method, we propose to address the structure-function relations within UCPs and MCU to understand the molecular mechanisms that govern H+ and Ca2+ translocation via these membrane transport proteins. For UCPs, we plan to identify the mechanisms by which free fatty acids, the endogenous UCPs activators, cause H+ translocation via UCPs. We also propose to develop a new generation of drugs that can activate H+ leak via UCP and be used for the treatment of type II diabetes, obesity, and fatty liver. For MCU, we propose to reveal the molecular mechanisms responsible for the exceptionally high Ca2+ selectivity of MCU, the MCU inward rectification (one-way permeation of Ca2+ into mitochondria), and the MCU potentiation by cytosolic Ca2+. This research will resolve several long-standing problems in the field of bioenergetics and will eventually enable pharmacological control of key mitochondrial functions in therapeutic and research purposes.